INCREASED AND PROLONGED STAT5 ACTIVATION DETECTED FOR A JAK2 E846D GERMLINE MUTATION IN THE SPECIFIC CONTEXT OF EPO RECEPTOR

Background

Somatic mutations of a gene that encodes the Janus kinase 2 (JAK2) are the most observed disease-causing events of the myeloproliferative neoplasms (MPN). Recently, germline JAK2 mutations have been described in cases with familial MPN, exhibiting hereditary thrombocytosis.

Aims
To characterize a novel JAK2 germline mutation found in a patient with erythrocytosis.

Methods

The effect of JAK2 E846D mutation was functionally analyzed using Ba/F3-EPOR cells (immunoblots, BrdU, MTT, inhibitors assays) and JAK2-deficient γ2A cells (reporter luciferase assay).

Results
The patient was diagnosed with increased red cell mass, facial plethora and splenomegaly at the age of 15. The bone marrow aspirate showed features typical for MPN. Two heterozygous mutations were found by targeted analyses; E846D in JAK2 inherited from the mother and Q157H in PHD2 (SNP rs61750991) inherited from the father. Erythroid progenitors derived from the patient and his parents were hypersensitive to erythropoietin (EPO), however only the patient exhibited the erythrocytosis phenotype. In silico modeling of JAK2 showed that the substitution of Glu by a residue one CH2 shorter, namely Asp, in the kinase domain might result in a formation of a tighter salt bridge to residue K926, more difficult to disrupt upon returning of JAK2 to its inactive state. This leads to a prolonged activation of JAK2 kinase.

Ba/F3 JAK2 E846D stable transfectants have revealed EPO-induced increased and prolonged JAK2 and STAT5 activation detected by immunoblots assessing active forms of JAK2 and STAT5. This was subsequently confirmed by luciferase assay in γ2A cells that detected higher and prolonged cytokine-induced levels of STAT5 transcriptional activity. Surprisingly, among the STAT family members, the E846D substitution significantly increased only STAT5 transcriptional activity and only in the presence of EPO receptor but not with TPO or G-CSF receptors. Two JAK2 inhibitors (Ruxolitinib, AZ-960) reduced the prolonged STAT5 activation; the E846D variant exhibited similar sensitivity as oncogenic JAK2 V617F. Although the E846D did not support growth factor independence of Ba/F3 cells as V617F, increased STAT5 activity was detected also in the condition without cytokines in transcriptional assays. In addition, Ba/F3 JAK2 E846D transfectants showed improved survival in EPO-limiting conditions when compared to JAK2 wild-type (wt) cells. Cell cycle studies demonstrated that upon growth factor starvation JAK2 E846D cells re-enter faster into S phase in low EPO concentrations, when compares to JAK2 wt.

Summary
The germline JAK2 E846D mutation emerged as the main disease causing event in our patient. It causes EPO hypersensitivity and erythroid hyperproliferation due to increased and prolonged JAK2/STAT5 signaling. This mutation gives this phenotype only in the context of the EPO receptor, explaining the erythrocytosis in vivo. We propose that the disease is a hereditary myeloproliferative disorder with incomplete penetrance; we observed full clinical phenotype in the patient and intermediate phenotype in the mother, the genetic background likely contributing to the extent of disease expression.

Grants support: P301/12/1503, NT13587-4, CZ.1.07/2.3.00/20.0164, CZ.1.07/2.3.00/30.0041 from Czech Republic; MEXP31C1, ARC10/15-027, FRIA PhD fellowship from Belgium.

Keyword(s): Erythrocytosis, Myeloproliferative disorder, STAT5

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